Fowl cholera is a severe respiratory disease in domestic poultry and wild birds. This disease occurs throughout the year and causes great losses in the poultry industry around the world (Rhoades et al., 1989). Fowl cholera has two clinical forms, acute disease and chronic disease. The acute disease is a septicemia with high morbidity and mortality. Clinical signs of the acute disease include fever, anorexia, ruffled feathers, mucous discharge from the mouth, diarrhea, increased respiratory rate and cyanosis. Death may be the first evidence of the acute disease. The chronic form of fowl cholera is characterized by localized infections including swelling in wattles, sinuses, periorbital subcutaneous tissues, leg or wing joints, sternal bursae and foot pads, exudative conjunctivitis, pharyngitis, emaciation and lethargy (Rhoades et al., 1991; Rhoades et al., 1989).
Pasteurella multocida is the causative agent of fowl cholera. Almost all types of birds including chickens, turkeys, ducks and geese are susceptible to P. multocida infection. In addition to causing fowl cholera in birds, P. multocida also causes disease in mammals, including cattle, swine, horses, sheep, goats, rabbits and humans. Differences in the capsule antigen are employed to classify P. multocida into serogroups, i.e., serogroups A, B, D, E and F (Carter et al., 1955; Rimler et al., 1987). Differences in lipopolysaccharide antigen are used to subtype the bacteria into 16 somatic serotypes (Brogden et al., 1978; Heddleston et al., 1992). Most of the avian isolates are serotype A: 1, A:3, and A:4 (Hofacre et al., 1986).
Current control of fowl cholera mainly relies on vaccination. Two kinds of vaccines are used in poultry, the inactivated vaccines (bacterins) and attenuated live vaccines. However, a bacterin induces only homologous, i.e., serotype-specific, protection. As there are currently 16 somatic serotypes, the efficacy of bacterins is very limited. While live vaccines such as those which employ the CU, PM-1 and M-9 strains, can induce heterologous immunity, they can induce disease (Carpenter et al., 1988; Hofacre et al., 1989a; Hofacre et al., 1989b; Hofacre et al., 1986; Schlink et al., 1987a; Schlink et al., 1987b).
The outer membrane of Gram-negative bacteria contains a number of components: phospholipid layer, outer membrane proteins (OMP), and lipopolysaccharide (LPS). The outer membrane contains a number of proteins including major outer membrane porins and other proteins. There are reports that P. multocida outer membrane protein was able to induce protective immunity in poultry and other animals (Lu et al., 1991a; Lu et al., 1991b; Lu et al., 1988a; Lu et al., 1988b; Ruffolo et al., 1996; Zhao et al., 1995). Moreover, it has been reported that outer membrane proteins expressed by in vivo grown bacteria induced cross-protection in poultry (heterologous protection) (Rimler et al., 1994; Wang et al., 1994a; Wang et al., 1994b).
Thus, what is needed is the identification and isolation of the gene encoding the major outer membrane protein of P. multocida. Moreover, there is a need for improved immunogenic compositions useful to prevent or inhibit fowl cholera.
The invention provides an isolated and purified nucleic acid molecule comprising a preselected nucleic acid sequence which encodes an avian Pasteurella multocida outer membrane protein or polypeptide (OmpH), a biologically active subunit or a biologically active variant thereof. Preferably, the nucleic acid molecule of theinvention encodes a porin. Porins are major outer membrane proteins which have pore-forming function. They serve as molecular sieves allowing polar solutes to pass through but excluding non-polar molecules of comparable sizes. As described hereinbelow, the genes (ompH) encoding OmpHs from all 16 P. multocida serotypes were cloned and sequenced. The OmpHs from different serotypes showed high homology in amino acid sequence (72.3% overall identity). Computer-aided secondary structure predictions revealed 16 anti-parallel beta-strands connected by 8 external loops and 8 short periplasmic turns. Preferred isolated nucleic acid molecules of the invention include those having a nucleic acid sequence comprising SEQ ID NO:1 or SEQ ID NO:3, molecules encode a polypeptide having an amino acid sequence such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 or SEQ ID NO:43. The nucleic acid molecules of the invention, subunits or variants thereof, are useful to prepare probes, primers or expression cassettes which, in turn, are useful to detect, amplify and express other ompH genes and related genes.
Therefore, the invention also provides an expression cassette comprising: a preselected DNA sequence which is operably linked to a promoter functional in a host cell, which DNA sequence encodes a Pasteurella multocida outer membrane polypeptide, a biologically active subunit or a biologically active variant thereof. The host cell may be prokaryotic or eukaryotic in origin. Preferably, the preselected DNA sequence comprises SEQ ID NO:1 or SEQ ID NO:3, or the complement thereto. Also preferably, the preselected DNA segment encodes a polypeptide having an amino acid sequence such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or a fragment thereof. These cassettes may be employed to prepare recombinant polypeptides. For example, a nucleic acid molecule of the invention, a variant or a fragment thereof, e.g., an expression cassette of the invention, may be introduced and expressed in a cultured host cell, preferably a host cell that is stably transformed with the nucleic acid molecule, so as to yield recombinant Pasteurella multocida outer membrane polypeptide, a biologically active subunit, variant or derivative thereof. Preferably, the recombinant polypeptide is recovered from the host cell. The recombinant polypeptide may be recovered from cell lysates in a soluble fraction, i.e., the recombinant OmpH is not associated with membranes, in the insoluble fraction, or from supernatants, i.e., the recombinant polypeptide is secreted into the extracellular environment of the host cell. As described herein, the genes encoding mature OmpH polypeptides of Pasteurella multocida strains X-73 and P-1059 were expressed as fusion proteins in Escherichia coli. Thus, another embodiment of the invention is a fusion polypeptide comprising at least an immunogenic or antigenic portion of the Pasteurella multocida outer membrane polypeptide.
Yet another embodiment of the invention is isolated and purified native Pasteurella mullocida outer membrane protein, which native polypeptide is obtained from a particular isolate or strain of Pasteurella multocida. As shown hereinbelow, the major outer membrane proteins of Pasteurella multocida strains X-73 and P-1059 were purified by selective extraction with detergents and size exclusion chromatography. The planar lipid bilayer assay showed that the isolated and purified native OmpH of X-73 and P-1059 had pore-forming activity. Thus, a preferred embodiment of the invention is isolated and purified Pasteurella multocida strain X-73 outer membrane protein, a biologically active subunit, variant or derivative thereof. Another preferred embodiment is isolated and purified Pasteurella multocida strain P-1059 outer membrane porin protein, a biologically active subunit, variant or derivative thereof.
The invention also provides an isolated and purified peptide of the Pasteurella multocida outer membrane polypeptide, a biologically active variant or derivative thereof. Preferably, a peptide of the invention corresponds to extracellular domains of Pasteurella multocida outer membrane polypeptide. More preferably, a peptide of the invention is modified so as to yield a derivative of the peptide, e.g., a cyclized peptide. A peptide or peptide variant of the invention is useful in assays to detect antibodies specific for the peptide or for a polypeptide, a portion of which has an amino acid sequence corresponding to an epitope within the peptide. A peptide or peptide variant of the invention is also useful in an immunogenic composition to prepare antisera or vaccines.
A vaccine of the invention comprises an amount of isolated and purified Pasteurella multocida outer membrane protein, polypeptide, a subunit, a fragment (e.g., a peptide), a variant, a derivative, or a combination thereof, effective to immunize a susceptible vertebrate, e.g., a bovine, swine, equine, ovine, caprine, rabbit, human or avian, preferably in combination with a carrier vehicle. Preferably, the polypeptide is the outer membrane polypeptide of strain X-73 or P-1059 of Pasteurella multocida. Protection studies described herein showed that isolated and purified X-73 outer membrane polypeptide induced homologous protection in chickens.
Also provided is a vaccine comprising an amount of a peptide of the outer membrane polypeptide of Pasteurella multocida, a variant, a derivative, or a combination thereof, effective to immunize a susceptible vertebrate, optionally in combination with a carrier vehicle. A peptide vaccine of the invention preferably provides homologous protection, and more preferably heterologous protection, i.e., protection against at least two different serotypes. Preferred peptides of the invention useful to prepare vaccines or immunogenic compositions correspond to extracellular sequences, e.g., from loop 2, of the outer membrane polypeptide. Protection studies showed that a synthetic cyclic peptide derived from the predicted largest loop (loop 2) of X-73 outer membrane polypeptide, i.e., a peptide comprising SEQ ID NO:45, induced homologous protection in chickens. In contrast, synthetic linear peptides of loop 2 and loop 5 of X-73 outer membrane polypeptide did not provide protection in chickens against homologous challenge. Thus, it is envisioned that a mixture of peptides, preferably cyclic peptides, may induce broad protection, i.e., heterologous protection, against Pasteurella multocida infection. Moreover, it is envisioned that any of the immunogenic compositions of the invention may be employed in combination with bacterins.
Further provided is an immunogenic composition comprising an effective amount of an isolated and purified Pasteurella multocida outer membrane polypeptide, e.g., X-73 or P-1059 polypeptide, a subunit, a peptide, a variant, a derivative, or a combination thereof, in combination with a pharmaceutically acceptable carrier, such as an injectable or ingestible liquid carrier, wherein the administration of the immunogenic composition to a vertebrate induces the production of antibodies specific for Pasteurella multocida outer membrane porin polypeptide.
The invention further provides a method for detecting or determining the presence antibodies which are specific for avian Pasteurella multocida in a vertebrate physiological sample. The method comprises contacting an amount of purified immunogenic protein (native), polypeptide (recombinant), peptide or variant thereof, encoded by the genomic nucleic acid of Pasteurella multocida with the physiological sample which comprises antibodies suspected of specifically reacting with Pasteurella multocida, for a sufficient time to form binary complexes between at least a portion of the antibodies and an antigenic or immunogenic portion of the purified protein or polypeptide. Then the presence or amount of the complexes is detected or determined. Alternatively, isolated cells of Pasteurella multocida, such as a lysate from, for example, strain X-73 or P-1059, can be employed as the immunogenic material that is contacted with the physiological sample to be tested. Thus, the invention also provides kits useful to detect or determine the presence of antibodies that specifically react with an infectious agent which is associated with fowl cholera. Such a kit may comprise packaging, containing, separately packaged: (a) a solid phase capable of immobilizing a polypeptide; and (b) a known amount of a purified recombinant polypeptide or peptide which specifically reacts with antibodies specific for the agent, wherein the amino acid sequence of the polypeptide or peptide is identical to at least a portion of the sequence of the outer membrane polypeptide of Pasteurella multocida, or a variant thereof.
Also provided is a method for detecting nucleic acid encoding an immunogenic polypeptide associated with the causative agent of fowl cholera. The method comprises contacting an amount of nucleic acid obtained from a vertebrate physiological sample which comprises cells suspected of containing nucleic acid encoding the immunogenic polypeptide, with an amount of at least two oligonucleotides under conditions effective to amplify the nucleic acid so as to yield an amount of amplified nucleic acid. Methods to amplify nucleic acid are well known to the art. A sample of RNA may be transcribed to DNA by reverse transcription of RNA from the physiological sample. At least one oligonucleotide is specific for the nucleic acid encoding an outer membrane polypeptide of Pasteurella multocida. The presence of the amplified nucleic acid is then detected or determined. Preferably, amplified DNA is subjected to agarose gel electrophoresis prior to detection. Thus, the invention also provides a diagnostic kit for detecting the presence of DNA associated with fowl cholera in a sample. The kit comprises packaging containing (a) a known amount of a first oligonucleotide, wherein the first oligonucleotide consists of at least about 7 to about 50, preferably at least about 12 to about 40 nucleotides, and even more preferably about 15 to about 25 nucleotides, and wherein the oligonucleotide has at least about 70% contiguous sequence identity to a nucleic acid molecule of the invention, e.g., SEQ ID NO:1, and (b) a known amount of a second oligonucleotide, wherein the second oligonucleotide consists of at least about 7 to about 50, preferably at least about 12 to about 40 nucleotides, and even more preferably about 15 to about 25 nucleotides, and wherein the oligonucleotide has at least about 70% contiguous sequence identity to a nucleotide sequence which is complementary to a nucleic acid mole cule of the invention, e.g., SEQ ID NO:1.
Yet another embodiment of the invention is a diagnostic method. The method comprises contacting an amount of DNA obtained from a physiological sample which comprises cells from a bird at risk of or afflicted with fowl cholera, or a vertebrate exposed to said bird, with an amount of at least two oligonucleotides that bind to complementary strands of said DNA at preselected regions under conditions effective to amplify the DNA, e.g., by a polymerase chain reaction, so as to yield an amount of amplified DNA. Alternatively, the DNA may be obtained by reverse transcription of RNA from the physiological sample. At least one of the oligonucleotides is specific for DNA encoding an outer membrane polypeptide of Pasteurella multocida. Then the presence of amplified DNA is detected or determined. The presence of amplified DNA is indicative of a bird infected with Pasteurella multocida, or afflicted with or at risk of fowl cholera, or a vertebrate exposed to said bird.
Yet another embodiment of the invention is an isolated antibody or a preparation thereof obtained from a vertebrate exposed to an immunogenic composition or vaccine of the invention wherein the preparation comprises antibodies that bind the infectious agent that is associated with fowl cholera. Preferably, the preparation is obtained from a chicken or a turkey. More preferably, the preparation binds to more than one serotype of the infectious agent. The preparations of antibodies of the invention are useful in assays, such as those described above, or to provide passive immunity to a vertebrate to which the preparation is administered.
Also provided is a method to prevent or inhibit fowl cholera, comprising: immunizing a bird with an effective amount of isolated or purified Pasteurella multocida outer membrane polypeptide, e.g., from strain X-73 or P-1059, a variant, a subunit (e.g., a peptide), a derivative, or a combination thereof. Preferably, the avian is immunized with a peptide of the invention, a variant, a derivative, or a combination thereof. Thus, the invention further provides an immunized bird, such as a turkey or chicken, i.e., that is resistant to infection by fowl cholera.